Tuesday, July 10, 2012

Precautions for Intravenous (IV) injection of Clodronate Liposomes

Clodrosome is usually dosed intravenously to mice and rats in a relatively large volume, or about 1% of their body weight. This, along with the fact that the Clodrosome suspension is slightly more viscous than water, increases the possibility of adverse reactions due to improper injection techniques. When injecting Clodrosome intravenously, the following guidelines will limit the potential for adverse reactions, including death. If the technician performing the injections is unfamiliar with injecting larger volumes (≥1% of the animals body weight) and/or viscous suspensions, it is useful for the technician to practice on non-experimental animals prior to initiation of the experimental protocol. Extra animals should be on hand should animals demonstrate suspicious behavior during or after Clodrosome treatment.

These precautions listed below apply to any intravenous injection method or site since the liposomes will always initially pass through the lung capillary bed before they enter the arterial circulation for distribution throughout the animal. Prior to its expiration date, uncontaminated Clodrosome (which has been maintained according to package instructions) is not toxic when dosed intravenously to animals according to the following guidelines. Animals expiring during the experimental protocol for reasons unrelated to the model should be necropsied and examined for opportunistic infections or other causes of premature death. Premature deaths, outside of injection-related adverse reactions, cannot be attributed to Clodrosome treatment alone.

Plan the experimental setup and protocol keeping in mind that the animals receiving Clodrosome will become immune-suppressed within hours after treatment making sterile procedures for dosing and handling the animals more important than usual.

While full-blown immunocompromised animal containment is usually not necessary (depending on the size and potential for contamination in the facility), extra precautions need to be implemented, such as more frequently supplying clean bedding, water-bottles and food on a daily basis, as well as closely monitoring the animals for signs of infection or illness not related to the experimental model.

Take into account that Clodrosome-treated animals may succumb to infectious disease models at a higher rate than normal animals requiring shorter intervals for assessing the animals’ conditions.

A significant number of unexpected deaths, outside of injection-related adverse reactions, should be investigated as it is likely due to opportunistic infections whose source must be eliminated from the facility environment or handling procedures.

Inflammation or necrosis at the injection site of only a few animals suggests contamination during the injection procedure while such reactions in several animals can be an indication that the clodronate liposome suspension has been contaminated. In this case the validity of the experiment should be seriously questioned.

Generally, the maximum tolerable volume (ml) dosed to animals in a single, intravenous bolus should be limited to approximately 1% of the animal’s body weight in grams.

For example, the maximum volume dosed to a 20 g mouse is 0.2 ml. Many sources will cite more conservative guidelines of 5 ml/Kg (0.1 ml/20 g mouse) or a maximum 4-5% dilution of the total blood volume (TBV~1.2 ml/20 g mouse X 5% = 0.06 ml/mouse), however, if the technician is experienced in maintaining a slow, steady injection rate along with the other precautions listed here, the larger volume is well tolerated.

In the event that the investigator suspects that injection-related adverse events are interfering with the study, Clodrosome can be diluted and dosed via intravenous infusion or smaller doses may be evaluated for effectiveness. However, this is rarely necessary.

Clodrosome should be at RT for dosing to healthy animals; infected or otherwise compromised animals (i.e. SCID or nude mice) may require Clodrosome be warmed to the animal’s body temp prior to injection.

Animals that are dosed intravenously with larger volumes of cold fluids can go into hypothermic shock.

Note that Clodrosome should be maintained at 4°C as much as possible. Consider making a sterile transfer of the total volume required for a single experiment from the original vial to another sterile container which can be warmed to the appropriate temperature, if the animal dosing protocol is expected to require Clodrosome to be maintained at elevated temperature for longer than an hour.

Immediately before extracting Clodrosome from the vial, visually inspect the suspension, making sure that it is homogeneous with no visible particulates.

Occasionally, liposomes will “settle” toward the bottom of the vial in which case the vial should be slowly and smoothly inverted several times.

Never vigorously shake liposomes; they will not “break”, but lipid-stabilized air bubbles can be introduced into the suspension. If these are transferred into the syringe and dosed to an animal, it could be seriously injured or killed by air emboli (air bubbles introduced into the bloodstream).

Also visually inspect Clodrosome once it is drawn into the syringe to ensure that no air bubbles have been introduced during its extraction from the vial. Following proper syringe-filling and air-bubble-removal procedures should prevent any incidents due to air emboli.

Some claim that air emboli are only an issue when introduced into arteries and that venous air emboli diffuse into the lungs when passing through the lung capillary bed, however many report adverse reactions when suspensions containing air bubbles are injected intravenously.

It is probable that air-bubbles in the Clodrosome suspension are stabilized by a monolayer of lipid, and therefore may not be as readily dissolved as air bubbles in an aqueous solution. This is characteristic of liposome suspensions and does not indicate a quality issue with Clodrosome. However, if a substantial increase in bubbles or foam is noticed with older Clodrosome suspensions, it may be an indication of degraded lipid and the Clodrosome should not be used.

The other important reason for removing all air bubbles is that included air bubbles occupy space in the needle and syringe so that the volume of suspension in the needle/syringe is less than the measured volume.

Syringes are calibrated to deliver the indicated volume with the syringe tip and needle hub completely filled.

It is not an issue when dosing Clodrosome directly from the vial, but the dead volume in a 1 ml syringe and needle hub is substantial (70 µl) compared to the 100-200 µl dose intended for mice. If this dead volume is not taken into account when using a single 1 ml syringe to draw up and/or dilute a suspension within the syringe, significant dosing errors can occur [11].

If necessary, a temporary engorgement of the vein may make the injection easier.

For example, some technicians briefly dip the tail into warm—not HOT—water in order to make the vein more prominent; warm compresses may be applied to rabbit ears; tourniquets are appropriate for use on limbs in dogs.

Some technicians place animals under heat lamps, but this is discouraged, especially in small rodents, as the animals are often overheated leading to additional trauma and discomfort.

If the vein is not easily visualized or accessed, consider using another injection route, such as retro-orbital injection as described above.

Remember that any change in protocol usually must be approved by the institutional animal care and use committee, therefore it is often useful to include alternative injection sites and/or methods in the initial application to the committee.

Once the needle is inserted into the tail vein, slowly and steadily inject the suspension at a rate of no more than 1 ml/minute. This translates to taking about 12-15 seconds to inject a 0.2 ml dose volume. Should the animal become overly agitated or begin gasping for air as discussed above, further decrease the injection rate. The slow injection rate allows the liposome suspension to be thoroughly mixed into the blood so that they are properly opsonized for optimal phagocyte uptake.

The animals should be closely observed for a few minutes for adverse reactions after the injection is completed and the animal returned to the cage. The investigator should consider excluding animals from the study which demonstrate significant aberrant behavior.